Metastasis is responsible for the majority of cancer-related deaths. A limiting step in metastasis is access to circulation, thus circulating tumor cell (CTCs) should be present in all patients with metastatic tumors, which may have both diagnostic and prognostic value. Accounting for only 3-5%, carcinoma of unknown primary (CUP) presents a challenge for clinicians in diagnosis and therapy. Tissue biopsy is the standard for diagnosis but there is need for improved methodology including a non-invasive blood test that relies on CTC analysis. Our overall objective is to utilize CTCs to provide insight into systemic cancer progression and approaches to therapy including a CTC blood test as a tool for diagnosis, prognosis and therapy. We hypothesize that real- time, single-cell immunophenotype profiling of CTC populations will inform diagnosis of tissue of origin, reveal prognostic markers such as stem-like properties and predict tumor aggressiveness. We have been developing a post-Veridex CTC analysis method using multiplexed Q-dot conjugated antibodies with the goal of detection of multiple markers in each single cell within a CTC population to aid in CUP diagnosis including tissue of origin. We have developed an immunofluorescence (IF) protocol with a minimal set of markers to predict the primary sites and provide prognostic information for common metastatic tumors: melanoma, lung, colorectal, and breast or prostate cancer. Two kits are available to isolate CTCs through Veridex CellSearch: melanoma kit (using melanoma antigen), and epithelial or carcinoma kit (using EpCAM). A patient's blood is initially tested with both kits, and positive CTCs from the melanoma kit supports a diagnosis of melanoma, whereas positive CTCs from the epithelial kit supports carcinoma. For carcinomas, we use cytokeratin 7 (CK7), cytokeratin 20 (CK20), thyroid transcription factor 1 (TTF-1), estrogen receptor (ER) or prostate-specific antigen (PSA) as the minimal set of markers. We are developing a flexible micro spring array (FMSA) size-based CTC enrichment protocol in parallel. IF is optimized in cultured tumor cells, first with individual antibodies, then quantum dots (Q-dot) conjugated multiplex quadra-antibody set. In addition, we have isolated viable patient-derived CTCs and maintained them in culture for a few weeks. Specific Aim #1 Develop a multiplex-marker based algorithm system for predicting primary sites in carcinoma of unknown primary. Specific Aim #2 Utilize the FMSA device and other approaches for isolating live CTCs to study the biological properties of these cells in the context of metastatic spread. Successful application of this protocol to CTCs in patient samples is expected to provide a noninvasive, relatively quick and inexpensive blood test that can be both diagnostic and prognostic, which may be a valuable adjunct to routine biopsy. Circulating tumor cells may provide insight into the mechanism of systemic cancer progression, the characteristics of metastatic founder cells and changes within these cells as cancer progresses, thereby impacting the efficacy and evaluation of cancer therapy.